Beta-lactamase variants
a betalactamase and variant technology, applied in the field of isolated polypeptides with betalactamase activity, can solve the problems of partial or complete loss of enzymatic activity, and achieve the effects of increasing activity, increasing enzymatic activity, and increasing activity
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example 1
tion of the Positions of Interest in the Sequence of VIM-2 Enzyme
[0078]In order to identify amino acid positions relevant for either the enzyme activity, its substrate profile, its stability or its level of production in the host cell, random mutagenesis was used to create a library of blaVIM-2 genes carrying up to 12 mutations. To this end, blaVIM-2 mutants were introduced using an error-prone polymerase chain reaction, in the presence of nucleotide analogues. The blaVIM-2-derived nucleotide sequences were then cloned in a suitable Escherichia coli plasmid vector and the properties of the enzyme carrying various substitutions (thus a laboratory variants deriving from the introduction of mutations in the blaVIM-2 nucleotide sequence) were analyzed using the evaluation of the beta-lactam susceptibility profiles of the strains producing the variants, the determination of the specific-hydrolyzing activity for the degradation of various beta-lactams and the stability of the variant enzy...
example 2
n and Purification of One VIM-2 Variant at One Position
[0080]The VIM-2 variant was produced in Escherichia coli using either a Plac-promoter-based system (using pLB-II high copy number plasmid, as described in Borgianni et al., Antimicrob. Agents Chemother.; 2010; 54:3197-3204) or a T7 promoter-based expression system (using the pET-9a expression plasmid). Briefly, the mutant blaVIM-2 gene was cloned in the plasmid vector pLB-II or pET-9a using the NdeI and BamHI restriction sites, and the resulting plasmid introduced in E. coli DH5α or BL21(DE3) cells by electroporation. The resulting host cell was grown in either Luria-Bertani medium or the rich auto-inducing cell culture medium ZYP-5052 (Studier, F. W. 2005. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41:207-234.) for 24 h, and the culture supernatant clarified by centrifugation. The resulting sample was then concentrated by ultrafiltration and loaded on an anion exchange chromatogr...
example 3
tion of the Increased Resistance to Protease for Variants of VIM-2 Enzyme
[0082]To measure the sensitivity of purified enzyme preparations (e. g. VIM-2, VIM-2 DCT236 and variants thereof, prepared as described in Example 2) to proteolytic degradation by commercially-available purified proteases, the enzyme (final concentration, 50 μg / mL) was incubated, at 35° C., for up to 150 min in a buffer (50 mM HEPES, 50 μM ZnSO4, pH 7.5) containing up to 2.5 mg / mL of trypsin (from bovine pancreas, Type III, Sigma-Aldrich Cat. No. T-8253). The residual activity of the resulting sample was determined by means of a spectrophotometric assay, in which the hydrolysis rate of a 150 μM imipenem solution (in 50 mM HEPES, 50 μM ZnSO4, pH 7.5) was measured and compared to that of the sample at the beginning of the incubation (time, 0 min) or in the absence of protease.
[0083]In these conditions and in the absence of trypsin in the buffer, both tested enzymes (VIM-2 and VIM-2 DCT236) incubated for up to 150...
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